Project Summary Chronic pain sufferers, an estimated one third of the American population, struggle to identify ways to reduce pain and improve their daily lives. Transient receptor potential (TRP) channels detect a wide range of physical and chemical stimuli, and through their integration of and response to these stimuli have an essential role in the pathophysiology of many chronic pain disorders. Notably, many natural products from plants and venomous animals target TRP channels, and can therefore be used to identify and characterize these important contributors to pain sensation. For instance, TRP melastatin 8 (TRPM8), the somatosensory receptor gated by cold temperatures, is also activated by natural cooling agents, such as menthol and eucalyptol, commonly used topical analgesic agents. Furthermore, TRPM8 is essential for the development of cold allodynia, a debilitating hypersensitivity to cold resulting from chemotherapy or other neuropathic insults. Despite the importance of TRPM8 and other TRP channels to the transition from acute to chronic pain states, the molecular basis for ligand binding, channel gating, and ion permeation remain incompletely understood. The objective of this proposal is to determine the mechanisms whereby TRPM8 conducts ions across cellular membranes in response to diverse signals, including those from plant-derived compounds that produce a cold sensation. The specific aims are to: 1) determine atomic structures of TRPM8 in different conformational states (K99 phase), 2) study the electrophysiological properties of TRPM8 (K99 phase), and 3) probe mechanisms of TRPM8 modulation by phosphatidylinositol lipids (R00 phase). Single-particle electron cryo-microscopy (cryo- EM) structures will be determined of TRPM8 alone and in complex with agonists, antagonists, or natural toxins. In particular, toxins from animal venoms are powerful tools for elucidating the structural mechanisms underlying channel gating and modulation. Structure-function analyses aimed at determining gating mechanisms and validating ligand binding sites will be conducted, as will biophysical studies of purified, reconstituted protein to functionally characterize intrinsic gating of TRPM8. Modulation by bioactive lipids is a unifying functional trait of TRP channels, including TRPM8, and thus effects of phosphatidylinositol lipids on TRPM8 will be explored and non-covalently bound lipids will be identified using native mass spectrometry. These goals are significant because they will enhance the biophysical and molecular understanding of pain sensation and, specifically, how TRPM8 modulation contributes to chronic pain. Ultimately, the aim is to assist in the rational design of novel TRPM8-based analgesic drugs. My mentor, Dr. Julius, as well as my expert advisors in cryo-EM (Dr. Cheng), electrophysiology (Dr. Kirichok), protein-lipid interactions (Dr. Marty), and pain signaling (Dr. von Zastrow), will provide training, in preparation for my career as an independent scientist.